Capped F10, a novel DNA directed fluoropyrimidine, in TP53 mutated acute myeloid leukemia (AML).
Authors: Timothy S. Pardee, Charles Okechukwu, Kristin Pladna, William Gmeiner
Journal: Journal of Clinical Oncology (2023)

7046 Background: Acute myeloid leukemia (AML) is an aggressive malignancy characterized by resistance to treatment and poor outcomes. One of the most devastating mutations in AML involves loss of the tumor suppressor TP53. AML harboring this loss carries a dismal prognosis and there is a clear need for new approaches. Methods: To identify new therapeutics, a TP53 null and WT isogenic cell line pair was generated using CRISPR/CAS9 using a murine AML cell line. Clonal isolates were obtained and loss of detectable TP53 was confirmed by Western Blotting. A high throughput screen of currently FDA approved anticancer agents was performed. Results: When agents were ranked by equal cytotoxicity against TP53 null and WT AML cells 20% (6/30) of the top 30 agents targeted the folate/thymidylate synthase axis. This suggests that thymidylate synthase (TS) is a vulnerability in TP53 null AML cells. To confirm TS as a target in TP53 null AML an additional murine AML TP53 null/WT cell line pair was generated using CRISPR/CAS9. When these cell lines were treated with doxorubicin the TP53 null cells were significantly more resistant (p≤0.001 at 10nM). Capped F10 (CF10) is a second generation fluoropyrimidine oligonucleotide that has been shown to be a highly potent inhibitor of TS. In contrast to doxorubicin, CF10 treatment resulted in significant cell death when dosed in the low picomolar range with minimal differences between the null and WT cell lines (p = 0.31 at 50pM). To extend this result TP53 WT or null cells were injected into syngeneic recipients and upon engraftment mice were treated with CF10 at 300mg/kg twice weekly. CF10 provided a significant survival benefit in the TP53 null but not WT injected mice consistent with CF10 having activity against TP53 null AML (p = 0.035 vs 0.13). To confirm a TP53 independent mechanism, competition assays were performed with CAS9 expressing AML cells partially infected with a vector expressing an sgRNA targeting TP53 and a GFP reporter. When the mixed population was treated with the IC 90 concentration of 5-fluorouracil there was a highly significant increase in GFP+ cells indicating a TP53 mediated cell death (p = &lt; 0.001). In contrast when cells were treated with the IC 90 concentration of CF10 there was a non-significant increase in GFP+ population (p = 0.074) consistent with a mainly TP53 independent mechanism. Fluoropyrimidine based therapy can be myelosuppressive especially in patients with low dihydropyrimidine dehydrogenase (DPD) activity. To assess the tolerability of CF10, healthy WAG/RAJ rats were treated with the DPD inhibitor ethynyl uracil at 2mg/kg alone or in combination with either 141 mg/kg CF10 or the molar equivalent of 50 mg/kg 5-flurouracil (5-FU) and weight determined. In contrast to 5-FU, CF10 treated animals did not have a significant change in weight compared to controls. Conclusions: Taken together these data suggest that CF10 is a promising new agent for TP53 null AML.